Aberration-correcting dark-field electron microscopy

ABSTRACT

A transmission electron microscope includes an electron beam source to generate an electron beam. Beam optics are provided to converge the electron beam. An aberration corrector comprising either a foil or a set of concentric elements corrects the electron beam for at least a spherical aberration. A specimen holder is provided to hold a specimen in the path of the electron beam. A detector is used to detect the electron beam transmitted through the specimen. The transmission electron microscope may be configured to operate in a dark-field mode in which a zero beam of the electron beam is not detected. The microscope may also be capable of operating in an incoherent illumination mode.

CLAIM FOR PRIORITY

This application is a continuation of application Ser. No. 13/024,961,filed Feb. 10, 2011, which claims priority under 35 U.S.C. §119(e) toProvisional Application No. 61/303,260, filed Feb. 10, 2010, andProvisional Application No. 61/352,243, filed Jun. 7, 2010, all of whichare incorporated herein by reference in their entireties.

TECHNICAL FIELD

This application relates to aberration-correcting, dark-field electronmicroscopy.

BACKGROUND

There are applications in which it is desirable to use electronmicroscopy to resolve a single point-like object in a specimen. Thesingle point-like object may be, for example, a single atom or a clusterof atoms on an amorphous substrate. Electron microscopy couldtheoretically be used to sequence bases of a nucleic acid, for example,such as the bases of a strand of deoxyribonucleic acid (DNA).

Scanning transmission electron microscopy (STEM), which raster scans anelectron beam across a specimen, can be used to resolve singlepoint-like objects in an image. However, STEM typically suffers from aslow scanning time, which causes poor throughput. For example, STEM mayinvolve scanning for a time on the order of 1 μs to 10 μs per pixel ofthe image. This scanning time may be inadequate where sequentialresolution of numerous single point-like objects is desired. STEMthroughput may be inadequate, for example, for sequencing a full humangenome in a practical amount of time.

Transmission electron microscopy (TEM), unlike STEM, images the specimenin parallel. But TEM imaging can be problematic when trying to resolvesingle point-like objects because the phase-contrast information istypically not directly interpretable for this purpose. For example, alight area in a TEM image could represent either an atom or the absenceof an atom. Accordingly, although TEM may have good throughput, it doesnot typically yield the desired information about the specimen.

Thus, it is desirable to have electron microscopy that can reliablyresolve point-like objects. It is further desirable for such electronmicroscopy to have substantially high throughput. Moreover, it isdesirable for such electron microscopy to be provided at an affordablecost.

SUMMARY

In one embodiment, a transmission electron microscope comprises anelectron beam source to generate an electron beam. Beam optics areprovided to converge the electron beam. The beam optics define an opticaxis of the microscope along which there is substantial cylindricalsymmetry of the beam optics. The microscope further comprises anaberration corrector comprising a foil located approximately at theoptic axis. The aberration corrector is adapted to correct the electronbeam for at least a spherical aberration. A specimen holder is providedto hold a specimen in the path of the electron beam. In addition, themicroscope comprises a detector to detect the electron beam transmittedthrough the specimen.

In yet another embodiment, a transmission electron microscope comprisesan electron beam source to generate an electron beam. Beam optics areprovided to converge the electron beam. The beam optics define an opticaxis of the microscope along which there is substantial cylindricalsymmetry of the beam optics. The microscope further comprises anaberration corrector comprising a set of elements approximatelyconcentric about the optic axis, the concentric elements being adaptedto have different respective electric potentials applied to them. Theaberration corrector is adapted to correct the electron beam for atleast a spherical aberration. A specimen holder is provided to hold aspecimen in the path of the electron beam. In addition, the microscopecomprises a detector to detect the electron beam transmitted through thespecimen.

In still another embodiment, a method is provided for diagnosingaberrations in a transmission electron microscope that is adapted tooperate in a dark-field mode in which a zero beam of the electron beamis not detected. The method comprises acquiring one or more images, as afunction of either illumination tilt or defocus, from the transmissionelectron microscope. A value is extracted for the blurring effect of thetilt or defocus from the images. A value is calculated for defocus orastigmatism based on the value for the blurring effect and the value foreither the illumination tilt or the defocus.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and aspectsof the transmission electron microscopes described herein and, togetherwith the description, serve to explain the principles of the invention.

FIGS. 1A and 1B are schematic diagrams of exemplary embodiments ofaberration-correcting ADF-TEM columns.

FIGS. 2A, 2B, 2C, 2D, 2E, and 2F are schematic diagrams of electronoptical rays in various exemplary embodiments of anaberration-correcting ADF-TEM column.

FIGS. 2G and 2H are schematic diagrams of an exemplary embodiment of afoil corrector of an aberration corrector.

FIGS. 3A, 3Ai, 3B, 3Bi, 3Bii, 3C, and 3Ci are perspective views ofvarious exemplary embodiments of charge-on-axis elements for anaberration-corrected ADF-TEM.

FIGS. 4A and 4B are schematic diagrams of exemplary embodiments ofcomponents for correcting parasitic aberrations in an ADF-TEM column.

FIG. 5 is a schematic diagram illustrating how the misprojection of thethird-order correction provides a fifth-order compensation.

FIG. 6 is a schematic diagram of an exemplary embodiment of anaberration-correcting ADF-TEM system capable of providing a mechanismfor aberration diagnosis.

FIG. 7 is a schematic diagram of an exemplary embodiment of a STEM modeof an ADF-TEM column.

FIGS. 8A, 8B, and 8C are illustrations of results of a computersimulation that was carried out to demonstrate the efficacy of anexemplary embodiment of a charge-on-axis aberration-correcting objectivelens assembly for use in an ADF-TEM.

FIG. 9 is a schematic diagram of an exemplary embodiment of animplementation of an incoherent illumination mode using an incoherentelectron source.

FIG. 10 is a schematic diagram of an exemplary embodiment of a referenceversion of an ADF-TEM.

FIG. 11 is a schematic diagram of an exemplary embodiment of animplementation in which image constituents from a tilted and scannedelectron beam are summed.

FIG. 12 is a set of plots illustrating a generalized version of themanner in which amplitude contrast is summed while phase contrast isdecreased to improve the image of an object being identified in thespecimen.

FIGS. 13, 14, 15, and 16 are schematic diagrams of various exemplaryembodiments of implementation of incoherent superposition.

DETAILED DESCRIPTION

A transmission electron microscope (TEM) is able to image a specimen inparallel, thereby theoretically offering rapid and efficient throughput.As explained above, however, TEM imaging can be problematic when tryingto resolve single point-like objects because the phase-contrastinformation in the image is typically not directly interpretable forthis purpose. This problem may arise, for example, when trying to imagesingle atoms or clusters of atoms in aperiodic arrangements on aspecimen.

TEM imaging can be adapted to operate in a “dark field” mode in which acentral beam (referred to as a “zero beam”) of electrons in the electronbeam of the microscope is blocked. Indeed, the dark-field mode may beimplemented as a primary or dedicated image mode for the TEM. Thedark-field mode can produce an image with monotonic contrast, whichallows direct interpretability of the image to determine relative atomicweights. For example, the dark-field imaging can be used to obtainchemically sensitive projections of single atoms, clusters of atoms, ornanostructures. However, the dark-field mode may decrease the datathroughput of imaging due to reduced electron dose, which taken alonemay be undesirable. Thus, dark-field imaging techniques based oncoherent illumination and suffering from spherical or other aberrationmay be undesirably slow.

In order to improve speed, TEM imaging may be adapted to correct foraberrations. Aberrations can be detected and a computer can be used toanalyze the aberrations and apply compensating signals toaberration-producing lens elements. The aberration correction canprovide increased throughput of imaging. Such increased throughput maybe especially advantageous in using TEM for DNA sequencing. The highthroughput may allow the microscope to be used in sequencing a fullhuman genome substantially quickly. For example, the microscope may beadapted to sequence a full human genome in from about 200 hours to about0.01 minutes, such as about 20 hours. In an especially high-throughputversion, the microscope may be used in sequencing a full human genome infrom about 10 hours to about 1 minute.

Thus, aberration correction may be implemented in a TEM that is alsoadapted to operate in the dark-field mode. As described further below,this combination may be especially advantageous when the aberrationcorrection is implemented wholly or in part using “charge-on-axis”elements. “Charge-on-axis” refers to one or more elements placedapproximately at the zero beam of the microscope. In a bright-fieldmode, in contrast, the zero beam would not be blocked by any suchelements.

In addition, and also as described further below, one or more of theaberration correction and the dark-field mode may be further combinedwith incoherent illumination. As described herein, incoherentillumination may be generated by, for example, the use of asubstantially incoherent electron source or shifting, scanning, oraltering the energy of the electron beam. The combination of aberrationcorrection, incoherent illumination, and dark-field operation mayespecially increase microscope throughput, at least in part due to anincreased electron dose from incoherent illumination as compared withcoherent illumination.

Reference will now be made in detail to exemplary embodiments of TEMs,which are illustrated in the accompanying drawings. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or similar parts.

In an exemplary embodiment of a TEM, an electron gun together withcondenser lenses and a pre-field of an objective lens form a patch ofelectron illumination on a specimen. The atoms of the specimen scatterthe incident electrons, with the heavier atoms scattering the electronsto higher angles. The post-sample field of the objective lens creates adiffraction pattern in a back-focal plane of the objective lens.

In an approximately cylindrically symmetric system, the dark-field modemay be an “annular-dark-field” (ADF) mode. In this case, an aperturecontaining a central beam stop may be mounted in or near the back-focalplane (or a plane conjugate to it). The central beam stop may have theshape of a circular disc. The beam stop limits the scattered electronsto an angular range denoted here as φ_(d), which defines an annulusbetween an inner angle φ₁ and outer angle φ₂. These angles may be, forexample, in the case of imaging clusters of atoms, from about 0.1 mradto about 10 mrad for φ₁, and from about 1 mrad to about 20 mrad for φ₂.In the case of imaging single atoms, these angles may be, for example,from about 5 mrad to about 20 mrad for φ₁, and at least about 20 mradfor φ₂. An example of a suitable range for φ₂ is from about 20 mrad toabout 50 mrad. Thus, an example of a suitable range for φ_(d) forimaging single atoms may be from about 15 mrad to about 50 mrad.Electrons passing through this annular aperture are ultimately collectedon a detector, generating an image of the specimen. In other words, therays passing through the annular aperture are ultimately the source ofthe information gleaned from the specimen.

The primary contrast mechanism for ADF-TEM may be mass-thicknesscontrast, providing enhanced sensitivity to atomic number of the imagedspecies. The collected intensity (I) may follow approximately therelation:I/I ₀=1−exp(−Nσρt)where N=N₀/A is Avogadro's number divided by atomic weight A; σ is theappropriate partial single-atom scattering cross-section dependent onatomic number Z, incident energy, and angular range; ρ is the materialsdensity; and t is the thickness.

The intensity increases monotonically with thickness, allowing readyidentification of relative atomic weights of species if thickness isconstant. While dark-field TEM is chemically sensitive, however, itsresolution may normally be compromised when used with conventionalelectron optical lenses. Since, in dark-field TEM, electron rays aremainly collected from higher angles, where the electron rays are morestrongly affected by aberrations, the contrast may become delocalizedand the resolution may be reduced. Thus, aberration correction may beespecially desirable when implemented in a dark-field-mode microscope.Furthermore, an aberration-correction implementation that appliescharge-on-axis to correct spherical aberration is inherently compatiblewith dark-field TEM because the beam stop of the aperture is located tointercept the path of rays proximal to the optic axis in the back focalplane of the objective lens.

An exemplary embodiment of the structural configuration of anaberration-correcting ADF-TEM is described below. This example of theaberration-correcting ADF-TEM has an electron optical column thatincludes an electron source, condenser lenses, a specimen holder, anobjective lens, and a detector. In certain embodiments of theaberration-correcting ADF-TEM, the electron source may be a thermionicsource, such as a tungsten (W) or lanthanum hexaboride (LaB₆) source.These sources may provide a substantially large current, which may beadvantageous in allowing shorter exposures for each image and hencehigher throughput. These examples of electron sources may not be ascoherent as other sources. High coherence levels are not necessarilyrequired, however, in aberration-correcting ADF-TEM. Indeed, significantadvantages can result from deliberately incoherent illumination, asexplained in more detail below.

The electron source may be followed by condenser lenses to form a beamthat will be incident on the sample. The condenser lenses may consistof, for example, two, three, or four lenses. The condenser lenses may bemagnetic or electrostatic. The electrons scattered from the specimen arethen imaged through an optical system. The optical system may accomplishat least two functions. First, the optical system may block out thecentral scattered beam to implement the ADF mode. Second, the opticalsystem may correct aberrations. The combination of these two featurescan be particularly advantageous.

The electromagnetic lenses can also include additional correctingelements near the axis. Furthermore, there is an opportunity forstandard magnifying lenses to be included in the aberration-correctingdark-field TEM. These magnifying lenses are followed by an electrondetector. The electron detector may have one of many forms that areknown to one of ordinary skill in the art.

The combination of the objective lens and the aberration-correctingsystem may be advantageous. The objective lens may structurally resemblea conventional electrostatic or magnetic lens. As part of theaberration-correcting system, a charge-inducing component may bepositioned at least partially on the optic axis of the ADF-TEM column(or a more conventional multipole-based aberration corrector), eitherbefore, in, or after the objective lens in relation to the beam path.

There exist alternative ways of introducing the charge-on-axis for theaberration-correcting ADF-TEM. For example, the charge-inducingcomponent may be constructed of metal serving to define anequipotential. Alternatively, the charge-inducing component may beconstructed of a substantially resistive material, such as a partiallyconductive material. In yet another embodiment, the charge-inducingcomponent is an insulator that is charged up in a particular way eitherby the electron beam itself or otherwise. The charge-on-axisconfiguration may allow the shape of the electric field to be controlledso that all of the rays emanating from one point in the object andpassing through this annular region are focused to a very tight spot inthe image plane.

The ADF-TEM may also include a system to correct for parasiticaberrations, in contrast to spherical aberrations, whether parasiticaberrations are cylindrically symmetric aberrations or not. Parasiticaberrations may be caused, for example, by the optical elements havingbeen machined in such a way as to be very slightly off-axis or veryslightly non-round.

An example of the correction of parasitic aberrations will now bedescribed. For low-order aberrations, namely up to second order, directcorrection by appropriate rotatable multipole or dipole alignment coilsmay be provided (e.g. quadrupole stigmator for astigmatism and sextupolefor threefold astigmatism). In a novel approach for mitigatingthird-order aberrations, the electron beam may be predistorted by amultipole element (quadrupole or octupole) before entering theaberration correcting element(s) and then undistorted by the samemultipole element after the aberration correcting element(s). Thisprocess can induce a non-round third-order aberration that corrects thenon-round parasitic aberration (two-fold symmetric in the case of aquadrupole (C32) and four-fold symmetric in the case of an octupole(C34)).

Fifth-order parasitic aberrations may be compensated by themisprojection of the correcting element(s) to an optical plane differentfrom the back focal plane of the objective lens. This would allowpropagation of the wave front to occur between the insertion of theaberration and its correction. This propagation causes a higher-ordercombination aberration whose sign depends on the sign of themisprojection.

This additional parasitic-aberration correction system can beadvantageous in many embodiments of aberration-corrected ADF-TEM,particularly in many commercial applications, because parasiticaberrations have been an important constraint on the development ofeffective aberration-correction systems for about 40 years. The abilityto correct these parasitic aberrations, therefore, may be asignificantly advantageous feature of aberration-corrected ADF-TEM.

The charge-on-axis implementation may enable miniaturization of thedark-field TEM, among several other advantages. In some applications,however, the advantages of miniaturization may not be required and otherconsiderations may even take precedence. Thus, alternatively tocharge-on-axis implementations of the aberration corrector, otherconfigurations of aberration correctors may be implemented.

In an example of such an alternative for an ADF-TEM, a standardaberration corrector may include a Nion Co. quadrupole-octupolecorrector or CEOS Co. sextupole or quadrupole-octupole corrector. Anannular aperture may be provided either in the incoming illumination ofthe sample (such as for a STEM mode of an ADF-TEM) or in the outgoingscattered beam (such as for an ADF-TEM mode).

The aberration-correcting dark-field TEM may additionally include amechanism for diagnosing the aberrations. Conventional approaches fordiagnosing aberrations typically assume that a bright-field image isavailable. One novel method for dark-field TEM is to acquire images as afunction of illumination tilt and defocus, and to extract the blurringeffect of the tilt and defocus. The blurring gives a value for thedefocus and astigmatism at a variety of angles. This process can providesufficient data to numerically compute an aberration function for theimaging system. A sample used for these purposes may contain singleatoms or clusters of atoms, or may be another kind of sample made forthe purpose of diagnosing aberrations. For example, the sample may bethe specimen that is ultimately the subject of interest for study.Alternatively, the sample may be a sample used solely for calibration ofthe aberration-correcting TEM.

FIG. 1A is a schematic diagram of an exemplary embodiment of anaberration-correcting ADF-TEM column 10. Column 10 has an electronsource 20, one or more condenser lenses 30, specimen 40, objective lens50, annular aperture 60, one or more projecting lenses 70, and detector80. A charge-on-axis aberration corrector 90 is incorporated inobjective lens 50. Image plane 100 is shown in the figure. Electronsource 20 and condenser lenses 30 are configured to provide a variety ofillumination conditions. For example, electron source 20 and/orcondenser lenses 30 may be adapted to provide a high-current,incoherent-illumination mode that achieves a unique synergy with theaberration-correction and ADF features of column 10.

Aberration-correcting ADF column 10 is connected to a power source thatprovides power to components of column 10, such as electron source 20,lenses 30, 50, and 70, aberration corrector 90, and detector 80, as wellas a stage that holds and moves specimen 40. Column 10 may have a totalpower consumption of less than about 800 W. In a low-power embodiment,such as where column 10 is miniaturized, column 10 may even have a powerconsumption of less than about 300 W, such as from about 10 W to about100 W. Electron source 20 may be adapted to generate an electron beamwith a current of less than about 100 mA. In an especially low-currentversion, electron source 20 may even be adapted to generate an electronbeam with a current of less than about 10 μA, such as less than about 10pA.

In aberration-correcting ADF-TEM column 10 of FIG. 1A,aberration-correcting objective lens 50 is internally corrected bycharge-on-axis corrector 90. In other words, the aberration correctionis performed inside the objective lens module. Locating charge-on-axiscorrector 90 inside objective lens 50 may be advantageous in reducingthe size of the EM column, which may otherwise be made unnecessarilylarge by an additional stack of corrective elements. Annular aperture 60may be inserted in or near the back focal plane of objective lens 50.Annular aperture 60 may also be positioned in any plane that isconjugate to the back focal plane of objective lens 50.

FIG. 1B is a schematic diagram of an exemplary variation of theembodiment of FIG. 1A, with charge-on-axis corrector 90 situated outsideof objective lens 50. This configuration can be constructed using, amongother parts, commercially-available components.

FIG. 2A is a schematic diagram of an exemplary embodiment of objectivelens 50 of the aberration-correcting ADF-TEM with charge-on-axisaberration corrector 90A, showing electron optical ray paths 110, 120.Parallel incident rays are shown in order to illustrate the effect ofthe aberration correction, although the lens would not typically beoperated with incident rays in parallel. In practice, objective lens 50is used to produce a magnified image of the illuminated specimen. Alsoshown are optic axis 130, central rays 110, peripheral rays 120,corrected central ray 140, corrected Gaussian focal plane 150, anduncorrected Gaussian focal plane 160. Lens elements 170 of objectivelens 50 are shown as schematic objects and represent, for example,either the pole-pieces and coils of a magnetic lens or the electrodes ofan electrostatic lens.

Charge-on-axis aberration corrector 90A is positioned along optic axis130. In one embodiment, objective lens 50 is disposed aroundcharge-on-axis aberration corrector 90A, such that charge-on-axisaberration corrector 90A is positioned between lens elements 170.Aberration corrector 90A provides a modified potential distribution thatcauses rays that are closer to the center of the beam, namely centralrays 110, to be focused to the same point as peripheral rays 120, whichare rays farther from the center of the beam.

Charge-on-axis aberration corrector 90A can be a metal cylinder, amongmany possible forms, connected to a power supply. Associated withcharge-on-axis aberration corrector 90A, there may be additionalelectrodes to establish the desired electrostatic fields. Thecombination of such an additional electrode and charge-on-axisaberration corrector 90A is referred to as a foil lens. When aberrationcorrector 90A is absent, the spherical aberration of objective lens 50causes rays that are near the center of the beam, namely central rays110, to be focused less strongly to second Gaussian focal plane 160,than rays far from the center of the beam, namely peripheral rays 120,which focus a shorter distance to first Gaussian focal plane 150.

Applying an electric potential to aberration corrector 90A provides alateral force to passing electron beam rays, causing the rays todeflect. This force more strongly affects rays passing closer to thecenter of the beam, causing them to focus nearer to the focal point thanrays far from optic axis 130. When peripheral rays 120 and central rays110 are focused to a common point by the combination of objective lens50 and charge-on-axis aberration corrector 90A, for example to firstGaussian focal plane 150, the effect of spherical aberration ismitigated or canceled.

FIG. 2B is a schematic diagram of an exemplary variation of the basicaberration-correcting ADF-TEM configuration shown in FIG. 2A. In thiscase, aberration corrector 90A is placed outside of objective lens 50.In this way, aberration corrector 90A does not interact with the fieldof lens elements 170. This positioning of aberration corrector 90Apre-aberrates central rays 110, as shown at the top of the figure. Inthis way, central rays 110 ultimately become focused to the same pointas peripheral rays 120.

The particular configuration of aberration corrector 90A shown in FIG.2B can allow peripheral rays 120 to focus approximately to theiroriginal focal point, although this is not necessarily required. Theconfigurations shown in FIGS. 2A and 2B affect peripheral rays 120 aswell as central rays 110, but in the proper proportions, causing them tofocus to the same point on Gaussian focal plane 150. This orientationand positioning of aberration corrector 90A thus affects central rays110 and peripheral rays 120, in the correct proportion and causes allthe rays to focus at a single point at Gaussian focal plane 150.Corrected Gaussian focal plane 150 may occupy any position after thelens.

FIG. 2C is a schematic diagram of an exemplary variation of the basicaberration-correcting ADF-TEM configuration shown in FIG. 2A, wherecharge-on-axis aberration corrector 90A is extended in the dimensionsperpendicular to optic axis 130, such that an element takes the form ofa foil, such as a conductive grid. This configuration of objective lens50 and charge-on-axis aberration corrector 90A can have approximatelythe same effect as the forms of charge-on-axis aberration corrector 90Apreviously depicted in FIGS. 2A and 2B. However, the alternativeconfiguration of charge-on-axis aberration corrector 90A shown in FIG.2C may be supported structurally more easily or in a more sturdy mannerthan separate aberration corrector 90A shown in FIGS. 2A and 2B. Thefoil form may be preferable for imaging point objects, in contrast togeneral imaging such as imaging of highly periodic objects. A foil formof aberration corrector 90A may be thin enough to be transparent to thecharged particles. Additionally, a grid may need to be fine enough sothat the distortion of the equipotential in each hole does notdetrimentally affect the resolution.

The metal used to produce the foil form of aberration corrector 90A maybe capable of being rendered sufficiently thin for this application. Anymetal foil may, in principle, be functional in thisaberration-correcting ADF-TEM column although there may be advantages tometals that do not scatter strongly and can be fabricated into thinfoils that are self supporting. A typical range for the thickness of thefoil is from about 0.1 nm to about 10 nm, such as from about 2 nm toabout 6 nm. For this reason, a wide variety of foil forms of aberrationcorrector 90A can be considered. A form can be selected among these tobest suit the anticipated needs of a particular application for theaberration-correcting dark-field TEM.

Virtually any foil can be selected, as well as alloys, such as forexample gold, silver, platinum, titanium, copper, and iron. Foils withhigher tensile strength can be employed for ease of manufacture androbustness in handling. In some cases, less expensive foil members canbe employed, depending on the intended application and the constraintsof the production budget. Ease of handling and resistance to materialfatigue may also be considered to limit production costs. The foil maybe so thin that it may be desirable to have the support of a metal gridthat acts as a scaffold. The ADF-TEM imaging mode may allow for theregion close to the optic axis to be used to provide robust mechanicalsupport without having a deleterious effect on the ADF-TEM image.

FIG. 2D is a schematic diagram of an exemplary variation of the foilform of aberration corrector 90A shown in FIG. 2C. In this case,however, the foil form is altered in shape to match the desiredmodification of the wave front to provide the desired aberrationcorrection. Again, the dark-field imaging mode may allow the center ofthe foil to be supported. Thus, the control of the geometry may be morepredictable than if the entire angular range needed to be as transparentas possible.

The result can be further improved by selecting the metal for the foil.For example, shape-retaining metal or transforming metal with shapememory can be employed. In this manner, the aberration-correctingADF-TEM can be used multiple times. A changed temperature can be used toreconfigure foil aberration corrector 90A to the shape needed for aparticular application. Thus, the aberration-correcting ADF-TEMconfigurations shown in FIGS. 2C and 2D could be switched betweenwithout having to manually substitute one of aberration corrector 90Afor the other.

In the case shown in FIG. 2D, a spherical aberration is to be corrected.There may be variants of each of FIG. 2C and FIG. 2D that are similar toFIG. 2B in that aberration corrector 90A is outside of objective lens50. FIG. 2E is a schematic diagram of such an exemplary variation of theembodiment of the aberration-correcting ADF-TEM shown in FIG. 2C. Inthis embodiment, the flat foil form of aberration corrector 90A isplaced outside of objective lens 50. FIG. 2F is a schematic diagram ofan exemplary variation of the embodiment of the aberration-correctedADF-TEM shown in FIG. 2D. In this embodiment, the curved or shaped foilform of aberration corrector 90A is placed outside of objective lens 50.

In one version, the foil form of aberration corrector 90A includes afoil across the beam path and an aperture in a different plane, the foiland aperture being adapted to receive independent electric potentials.FIGS. 2G and 2H illustrate top and side views, respectively, of theconstruction of an exemplary embodiment of a foil corrector. This foilcorrector includes metal layers 171 and 173, an insulating layer 172 anda thin conducting electron-transparent foil 174. Aperture 175 is alsoshown in the figure. Metal layers 171 and 173 are made of a metal, suchas for example copper. Insulating layer 172 may be a thick quartz sheet.In one example, insulating layer 172 has a thickness of about 50micrometers. During fabrication, insulating layer 172 may be preparedwith standard lithographic techniques so as to leave two alignedapertures, one on either side of the quartz sheet. Metal layers 171 and173 may then be deposited on insulating layer 172. A thin film of carbonmay then be deposited on metal layer 173 and exposed insulating layer172. A vapour acid etch (such as hydrofluoric acid) can then be used togently remove a portion of insulating layer 172 through the uncoveredaperture. This etch leaves a free-standing film of carbon that isextremely thin and therefore fulfills criteria of beingelectron-transparent and consisting of low-Z elements that may bedesirable for foil 174.

Both metal layers 171 and 173 and foil 174 may have openings withdiameters of from about 1 μm to about 100 μm. Insulating layer 172,meanwhile, may have an opening with a diameter of from about 1 μm toabout 600 μm. Where foil 174 spans aperture 175, foil 174 may have athickness of from about 5 nm to about 1000 nm. Alternatively, foil 174may be as thin as a monolayer of suitable material, such as graphene. Inthis latter case, insulating layer 172 may have a thickness of fromabout 1 μm to about 1000 μm while metal layers 171 and 172 may have athickness of from about 1 nm to about 1000 μm.

Each of the configurations shown in FIGS. 2B-2H ideally focuses electronbeam rays that emanate from one point on a specimen down toapproximately one point in image plane 160 formed by objective lens 50.

FIG. 3A is a perspective, simplified view of an exemplary embodiment ofan element 180 of charge-on-axis corrector 90A of theaberration-correcting ADF-TEM. In this embodiment, the charge regionsare continuous and arranged in one monolithic piece. The materials usedto produce element 180 can be selected using the constraints that thematerials must be robust enough mechanically and in terms of resistanceto the electron irradiation, have a conducting surface (or near surfacelayer), and be amenable to fabrication into the desired geometry. Thismay include metals such as platinum, titanium, molybdenum, and gold, butalso semiconductors like doped silicon and gallium arsenide. It wouldalso include composite structures of an insulator, such as siliconnitride that is coated with a thin metal layer.

The size of element 180 may range from about 1 nm to about 10 cm indiameter, with an overall depth ranging from about 10 microns to a fewcentimeters. The conductivity of the surface of element 180 may besufficient such that, when element 180 is connected to the microscopeground, there is substantially no evidence in the image that theresistivity of element 180 is causing time-dependent changes in theimage.

FIGS. 3A, 3Ai, 3B, 3Bi, and 3Bii are perspective, simplified views ofexemplary embodiments of various approaches to configuring electrodestogether to form element 180 of charge-on-axis corrector 90A. Theseconfigurations of charge-on-axis element 180 achieve different fielddistributions in the vacuum where the electrons travel. Embodiments ofelements 180 in these figures may need to be mechanically supported,such as shown in FIG. 3C. All of the geometries shown may optionally beaugmented by a thin resistive foil (such as a few nanometers thicknessof amorphous carbon) in a plane perpendicular to the optic axis andmechanically supported by charge-on-axis element 180 to furtherconstrain the shape of the equipotentials and thus more preciselyachieve the desired particle trajectories. In one version,charge-on-axis corrector 90A is made up of a total of from 1 to about 20distinct parts. In a more particularized version, charge-on-axiscorrector 90A is made up of a total of from 2 to 3 distinct parts.

FIG. 3Ai illustrates a variant of the aberration-corrected ADF-TEM inFIG. 3A in which element 180, while still a cylinder in total form, ismade up of multiple charge regions that are concentric. In this example,three charged regions 190 are separated by two insulators 200. Differentcharge values are placed on each of different charge regions 190.

FIG. 3B is a variant on the aberration-corrected ADF-TEM embodimentshown in FIG. 3A in which monolithically configured charge-on-axiselement 180 is cylindrically symmetric but has a shaped profile, in thiscase a straight line.

FIG. 3Bi illustrates a variant of charge-on-axis element 180 shown inFIG. 3B with the multiple charge region features of FIG. 3Ai. This takesthe form of three nested cones of diminishing sizes with differentvoltages.

FIG. 3Bii illustrates a variant of charge-on-axis element 180 shown inFIG. 3Bi. In this case, charge-on-axis element 180 has curved, ratherthan straight, sides. Again, charge-on-axis element 180 has a coaxialform, with multiple charged conductors 190 and insulators 200therebetween.

FIG. 3C illustrates yet another variant of charge-on-axis element 180,this time having concentric distributed charges on individual rings 210suspended in space by a web of spoke supports 220. Individual rings 210radiating out from the optic axis having different electric potentialvalues on them to produce a distribution of charges about the opticaxis. One or more of spoke supports 220 may additionally be used todeliver charges to each of independent rings 210.

Spoke supports 220 may be constructed of a conductor, such as metal, oran insulator. For metal, the electric potential on spoke supports 220may be of one value the entire way along the length of the spoke. Ifspoke supports 220 are constructed of an insulator material, on theother hand, spoke supports 220 may charge up. In a preferred embodiment,spoke supports 220 include insulators (which may surround a conductor)coated with another layer of material through which a current can flowdown the potential gradient to establish a desired shape of theequipotentials.

These configurations provide other possibilities for providingcharge-on-axis. Another way of introducing the charge-on-axis is anelectrostatic mirror. However, the electrons may come to a standstillusing this system after being decelerated by the field. As a result, theelectrons may become very sensitive to stray fields.

An oblique electrostatic mirror may be another way of providingcharge-on-axis aberration correction.

FIG. 3C illustrates several concentric electrodes centered on the opticaxis. This construction may resemble a zone plate. In this case,piecewise aberration correction but not complete aberration correctioncan be performed such that only correction of rays with large angles isachieved. In this case, a number of electrodes are provided with gapstherebetween. In each of the gaps, the local field provides aberrationcorrection to the rays passing through the gap. The result is that atleast a substantial fraction of the electrons passing through these gapswill contribute positively to the intensity of the image.

FIG. 3Ci illustrates an aberration-correcting ADF-TEM embodiment adaptedto provide a shaped potential distribution using a single power supply.One example of a solution is to provide a central charge-on-axis element225 supported by spoke supports 220 that are electrically conductive andalso have a coating 230. Coating 230 may be made of a resistor that isspecifically graded or has a changing thickness so that the electricpotential drops in a functionally correct way as a function of radialdistance from central charge-on-axis element 225 to achieve the desiredelectric potential distribution, as shown by equipotential lines 255 inthe figure. Alternatively, coating 230 may be made of a conductor toachieve a similar effect.

Spoke supports 220 in many embodiments of the aberration-correctingADF-TEM may be substantially complex. For example, spoke supports 220may contain a conductor 240 in the middle, an insulator 250 outside ofconductor 240, and then coating 230 on the very outside as shown in theexploded cross-section in FIG. 3Ci. This combination of conductor 240,insulator 250, and coating 230 may advantageously be used to provide thecorrect electric field distribution.

This approach to correcting aberrations in the aberration-correctingADF-TEM may also be used to correct for parasitic aberrations. Forexample, there exists an aberration referred to as the C32 aberrationthat comprises a lack of cylindrical symmetry of spherical aberration.For the sake of reference, a pure spherical aberration, such as the C30aberration, is cylindrically symmetric. The C32 aberration, however,describes how an otherwise spherical aberration (in the case of C30) istwo-fold symmetric. To correct the parasitic aberration referred to asthe C32 aberration, a multipole lens called a quadrupole may beinstalled before aberration corrector 90A. An additional quadrupole lensmay be installed after aberration corrector 90A.

The effect of this C32-correcting configuration is to pre-distort theelectron beam. In this case, the beam goes through the correcting lens,and the main distortion is eliminated after the correcting lens, leavingbehind an approximate ellipticity to the spherical aberration. Thissystem modification provides a C32 correction and a round beam.

To correct C34 aberration, or four-fold astigmatism, octupole lenses canbe employed in the same manner, namely one before and one afteraberration corrector 90A. For example, FIG. 2A shows sphericalaberration corrector 90A between lenses 170. Many configurations arepossible for the octupole lens. For example, the octupole lens may bepositioned before lens elements 170, between lens elements 170, or afterlens elements 170, similarly to charge-on-axis aberration corrector 90Ain FIGS. 2A and 2B, which could be above or below lens elements 170.

FIG. 4A is a schematic diagram of an exemplary embodiment of componentsfor correcting parasitic aberrations in an ADF-TEM column. In additionto basic aberration corrector 90A described above, additional multipoleparasitic-aberration correcting elements 260 can be positioned beforeand after lenses 170. This configuration may be provided to correctparasitic aberrations.

Additional aberration-correcting elements 260 can be very important inmany embodiments of the aberration-corrected ADF-TEM system, particularin many commercial applications. These parasitic aberrations heldaberration correction back for about 40 years; thus, the ability tocorrect parasitic aberrations may be a significant advantage of thepresent aberration-correcting ADF-TEM. The aforementioned approachesprovide examples of corrections to the third-order parasiticaberrations. The remaining off-axial aberration, namely coma, may beeliminated by a straightforward dipole shift of the beam with respect toa lens.

FIG. 4B is a schematic diagram of an exemplary embodiment of componentsfor correcting fifth-order parasitic aberrations in anaberration-correcting ADF-TEM column. In this fifth-orderaberration-correcting configuration, additional projecting lenses 270are situated between charge-on-axis corrector 90A and lenses 170.

Projecting lenses can also be utilized in an additional approach. In thecase of a negative third-order function and a positive third-orderfunction, these functions may substantially cancel each other. Howeverif there is a negative third-order component, and it is allowed topropagate for a distance and then cancel, the result is that the primarythird-order component cancels, and a fifth-order component occurs. Bydetermining the sign of that distance, the fifth-order aberrations canbe controlled.

FIG. 5 is a schematic diagram illustrating how the misprojection of thethird-order correction provides a fifth-order compensation. Whenwavefronts 280 and 290 with third-order shaping of opposite sign aresuperimposed in the same plane, they cancel and generate a flatwavefront 300. However, when a wavefront with positive third-ordercurvature 310 is allowed to propagate over drift distance d and is thenshaped using a negative third-order curvature 320, the superposition isa wavefront with negative residual fifth-order shaping 330. Analogously,when a wavefront with negative third-order curvature 340 is separated bya drift distance d from a positive third-order curvature 350, awavefront with positive residual fifth-order shaping 360 is generated.

FIG. 6 is a schematic diagram of an exemplary embodiment of anaberration-correcting ADF-TEM column capable of providing a mechanismfor aberration diagnosis. Illumination tilt 370 is provided prior tospecimen 40. For example, the beam may be tilted by two pairs ofsuitably placed and excited dipole deflectors (either magnetic orelectrostatic) (not shown). An image may then be acquired in the ADF-TEMmode in focus, under-focus, and over-focus for each of a variety oftilts. This approach provides an image that is relatively unblurred, aswell as two images that have known blurring. Afterward, the blurringfunction is deconvolved out. The results of these measurements providethe local defocus and astigmatism at each tilt angle. From this data,the aberration function can be deduced.

The various methods described above provide for correction of the mainspherical aberration, which is third-order, as well as correction of theparasitic aberrations and some control of the fifth-order aberrations.In total, these innovative methods of aberration correction provide afinely focused ADF-TEM image, by providing both a means for measuringthe aberrations and an apparatus for applying aberration correction.

The electron microscope described may also be configured in a scanningtransmission electron microscope (STEM) configuration to achieve asimilar imaging outcome. FIG. 7 is a schematic diagram of an exemplaryembodiment of a STEM mode of the aberration-correcting ADF-TEM that canbe implemented by the assembly of commercially available components.This STEM mode is formally optically equivalent pixel by pixel toaberration-correcting ADF-TEM 10 illustrated in FIG. 1A by the principleof reciprocity. In the ADF-STEM mode, source 400 corresponds to TEMdetector 80 in FIG. 1A, and similarly the detector 430 corresponds toTEM source 20 in FIG. 1A. The electron beam travels upward from source400 through condenser lenses 30 and passes through annular aperture 60,corrector (not shown), and objective lens 50. Objective lens 50 forms acollimated hollow conical probe on sample 410. The electrons thenscatter from sample 410, forming a diffraction pattern that may becollected by a projection system 70 that can be used to adjust themagnification of the diffraction pattern onto detector 430. Detector 430comprises a set of concentric rings and a center detector to collectrays scattered to different angles and provide an intensity signal. Scancoils 420 are excited with ramp waveforms, causing the collimated probeto be scanned across the sample and thereby producing an intensitysignal at the detector unique to the location of the probe on thesample. Descan coils 380 may be used to de-scan the beam symmetricallyto the scan coils in order to restore the diffraction pattern to theoptical path and prevent distortion. In this case, incoherence isprovided by a large on-axis detector (this detector would be abright-field detector if there were any bright-field electrons todetect). Although it is likely to be slower at collecting images, thisSTEM mode will have other advantages such as enabling simultaneous STEMADF-TEM, a type of annular bright field and a very high-angle annulardark field with hollow cone illumination. A high-brightness gun mayallow this mode to operate faster.

The features described herein for the aberration-correcting dark-fieldTEM may be implemented in many different types of microscopes utilizingcharged ions or other particle beams. Moreover, theaberration-correcting dark-field TEM may be used in any suitablefacility in any desired arrangement, such as networked, direct, orindirect communication arrangements.

Furthermore, the aberration-correcting dark-field TEM system may includeany quantity of components, such as microscopes, machine managers,computer systems, networks, and image stores, that may be incommunication with or coupled to each other in any suitable fashion,such as wired or wireless, over a network such as WAN, LAN, or Internet,directly or indirectly coupled, local or remote from each other, via anycommunications medium, and utilizing any suitable communication protocolor standard.

The embodiments of aberration-correcting dark-field TEM described hereinmay be implemented with either electrostatic or magnetic components. Forexample, for a commercial setting, a relatively small electrostaticversion of the aberration-correcting dark-field TEM may be constructed.The aberration-correcting dark-field TEM system may include any quantityof electrostatic or magnetic components, such as electron or otherparticle gun, lenses, dispersion device, stigmator coils, reflected anddischarged electron detector units, and stages, arranged within orexternal to the aberration-correcting dark-field TEM in any suitablefashion. Image stores, files, and folders used by theaberration-correcting dark-field TEM system may be of any quantity andmay be implemented by any storage devices, such as memory, database, ordata structures.

The aberration-correcting dark-field TEM may include a controller. Thecontroller may include any one or more microprocessors, controllers,processing systems and/or circuitry, such as any combination of hardwareand/or software modules. For example, the controller may be implementedin any quantity of personal computers, such as IBM-compatible, Apple®,Macintosh®, Android™, or other computer platforms. The controller mayalso include any commercially available operating system software, suchas Windows, Operating System/2®, Unix®, or Linux, or any commerciallyavailable and/or custom software such as communications software ormicroscope monitoring software. Furthermore, the controller may includeany types of input devices such as a touchpad, keyboard, mouse,microphone, or voice recognition.

The controller software, such as a monitoring module, may be stored on acomputer-readable medium such as a magnetic, optical, magneto-optic, orflash medium, floppy diskettes, CD-ROM, DVD, or other memory devices,for use on stand-alone systems or systems connected by a network orother communications medium, and/or may be downloaded, such as in theform of carrier waves, or packets, to systems via a network or othercommunications medium.

The controller may control operation of the aberration-correctingdark-field TEM column. Alternatively or in addition, the controller mayreceive an image from the detector of the TEM to be processedcomputationally. For example, the controller may process collectedparticle data and/or process any desired images. The controller mayinclude an image formation unit for this purpose. The image formationunit may be disposed within or external of the aberration-correctingdark-field TEM column and communicate with the microscope column in anyfashion such as directly or indirectly coupled, or communicate via anetwork.

Moreover, the various functions of the aberration-correcting dark-fieldTEM may be distributed in any manner among any quantity such as one ormore of hardware and/or software modules or units, computer orprocessing systems or circuitry, where the computer or processingsystems may be disposed locally or remotely of each other andcommunicate via any suitable communications medium such as LAN, WAN,Intranet, Internet, hardwire, modem connection, or wireless. Thesoftware and/or algorithms described above may be modified in any mannerthat accomplishes the functions described herein.

The aberration-correcting dark-field TEM may use any number of images ofa sample to determine optimal beam parameter settings and/or imagequality values. The images may cover any desired variation range for aparticular parameter. The sample may be of any quantity, may be of anyshape or size, and may include any desired features. For example, thesample may include a specific configuration for a desired application orparameter setting. The sample may be disposed at any desired location onor off the stage to acquire images. In one example, the sample is in theform of a product specimen such as a semiconductor device.

The aberration-correcting dark-field TEM may also use any number ofimages for the image quality comparison, where the image quality valuesfor current and prior images may be combined in any suitable fashion,such as averaged, weighted, or summed. The user threshold may be set toany suitable values depending upon the desired image quality. Thecomparison of image quality values may utilize any mathematical orstatistical operations to determine image quality compliance such as acomparison, statistical variance, or deviation.

The aberration-correcting dark-field TEM may analyze any suitablecharacteristics, such as intensity, pixel counts, or power, and utilizeany differentiating characteristic between settings in any desiredregion. The region of separation may be of any shape or size and belocated within any desired range. The aberration-correcting dark-fieldTEM may also utilize any suitable modeling or approximation techniquesto determine best fit lines and/or curves such as linear or non-linearregression, curve fitting, least squares, or integration. The models mayapproximate the data within any suitable tolerances. Theaberration-correcting dark-field TEM may identify any quantity ofseparation regions and utilize any suitable techniques to combine and/orselect resulting slope values such as lowest slope, average, weighting,or sum.

The parameter determination may be triggered in any suitable fashion.For example, the machine manager may monitor the microscope to initiatethe determination, the computer system or controller may periodicallyretrieve images based on a periodic acquisition of sample images or pollthe image store to determine the presence of sample images, or manuallytrigger determination. The quality inspection and/or parameterdetermination may be initiated in response to any suitable conditions(e.g., within any desired time interval such as within any quantity ofhours or minutes, subsequent any quantity of images generated by themicroscope such as every Nth scan performed by the microscope,subsequent any quantity of quality inspections.

The aberration-correcting dark-field TEM technique may be performedautomatically, where parameters are determined and applied to themicroscope. Alternatively, any part of the technique, such as scanningof images, determination of parameters, or application of theparameters, may be performed manually. For example, the computer systemmay provide the optimal settings to a technician that manually appliesthe settings to the microscope. The microscope controller may performany desired processing, such as monitoring and parameter adjustment orimage formation and processing.

Implementation of aspects of the aberration-correcting dark-field TEM,such as the image processing or aberration correction, may bedistributed among the computer system, microscope controller, or otherprocessing device in any desired manner, where these devices may belocal or remote in relation to one another. The computer system andmicrocontroller communicate with and/or control the microscope toperform any desired functions, such as scan the specimen and generatethe images or transfer images to memory.

A computer simulation was carried out to demonstrate the efficacy of anexemplary embodiment of an aberration-correcting ADF-TEM. FIGS. 8A, 8B,and 8C illustrate the results of this simulation providing preliminaryperformance results of a prototype charge-on-axis corrector. FIG. 8Ashows two points separated by 1 nm that are projected from image plane431 through unipotential lens 432, which exhibits spherical aberration.Unipotential lens 432 focuses the rays emanating from the two points toa beam crossover region 436 farther down the optical path. Aberrationcorrector 433 contains two charge elements 434, on which a thin foil 435is supported. The rays passing through the thin foil 435 are divergeddepending on their angle, correcting the spherical aberration inherentin unipotential lens 432. FIG. 8B shows a magnification of 150 times ofcrossover region 436, and FIG. 8C shows a magnification of 22,500 timesof the same crossover region 436, showing two distinct highly localizedcrossover points separated by distance d=14 nm, corresponding to amagnification factor by the corrected lens of 14 times. This simulationshowed a high degree of convergence by a charge-on-axis aberrationcorrector inside the objective lens.

This computer simulation also shows an embodiment of a noveldouble-sided foil corrector that is additionally asymmetric. Thedouble-sided corrector may allow the aberration corrector to achieve therequired amount of correction with smaller potentials than may berequired for a single-sided corrector. In addition, the asymmetry of thecorrector may allow some—or even complete—cancellation of fifth-orderaberrations by a mechanism similar to that shown in FIG. 5.

Aberration-correcting dark-field TEM enables high-throughput,atomic-resolution electron microscopy at a relatively small size. Inparticular, there may be a number of advantages to making theaberration-correcting dark-field TEM column small and usingelectrostatic components, as in one version. For example, such a columnmay be less expensive to construct because the components can berelatively simple. The components may include conductive electrodes thatare made of platinum, or alternatively metals such as platinum-coated orgold-coated aluminum molybdenum, titanium, or stainless steel. Althoughplatinum may be functionally advantageous, these alternative materialsmay make the commercial device substantially less expensive tomanufacture.

Aberration-correcting dark-field TEM can provide advantages in size,speed, and sensitivity over conventional electron microscope (EM)systems. With these advantages, new products and applications can bemade available to both research and commercial sectors. For example,aberration-correcting dark-field TEM can enable practical,high-throughput DNA sequencing, as described in more detail below.Moreover, very small EM structures may be provided, also as described inmore detail below. In addition, the aberration correction approachesdescribed above create a foundation for promising applications.

An example of an advantageous commercial embodiment ofaberration-correcting ADF-TEM is a small, relatively inexpensive column.For example, the column of the microscope may have dimensions of fromabout 2.0 m×1.5 m×1.5 m to about 10 cm×5 cm×5 cm, or the volumetricequivalent thereof. In an even smaller version, the column may havedimensions of from about 75 cm×50 cm×50 cm to about 10 cm×5 cm×5 cm, orthe volumetric equivalent thereof. The combination of small form-factorand low cost opens up the opportunity for entirely new, consumer-focusedapplications.

Beyond the cost and size advantage of this consumer-product approach todeveloping EM systems, there is also an advantage in the scaling of theoptics. Since optical properties scale with the physical size in anoptical system, the smaller the size of the aberration-correctingADF-TEM system, the smaller the aberrations. Thus, when theaberration-correcting ADF-TEM column is reduced in size, the resolutionimproves by the same factor up to the diffraction limit of the electronsat the operating energy. For example, the microscope may have a spatialresolution of from about 10 nm to about 0.01 nm. A preferable versionmay have a spatial resolution of from about 1 nm to about 0.1 nm.

A particularly useful application of aberration-correcting dark-fieldTEM is to analyze a DNA sample in order to determine the sequence of itsbase pairs. In one version, a single strand of DNA is stretched usingtechniques that have been described in PCT Publication No. WO2009/046445 dated Sep. 4, 2009, entitled “Sequencing Nucleic AcidPolymers with Electron Microscopy,” and filed as InternationalApplication No. PCT/US2008/078986 on Jun. 10, 2008 (this PCT publicationis incorporated herein by reference in its entirety). A particular setof bases has been labeled with a label that contains at least one heavyscatterer, such as a single heavy atom or a cluster of atoms. Examplesof such labels include osmium, triosmium, and platinum.

In a DNA sample labeled in this manner, the labels, such as single atomsor small clusters of atoms, scatter electrons strongly in an environmentthat otherwise includes elements that are light scatterers such as, forexample, carbon, hydrogen, and oxygen, with a few other elements, suchas a small amount of phosphorus, a small amount of nitrogen, and smallamounts of other relatively light elements. Therefore, the background onwhich the heavy scattering elements are to be measured by theaberration-correcting dark-field TEM may provide a good contrast to thelabels.

In this way, the combination of the dark-field mode and aberrationcorrection enables high-contrast imaging that permits the distancesbetween the labeled bases to be evaluated quickly. Theaberration-correcting dark-field TEM provides sufficient resolution tobe able to see the single atoms or clusters of atoms, given theresolution and the signal-to-noise ratio. The dark-field modediscriminates the heavier elements that scatter to higher angles fromthe lighter elements that scatter to lower angles.

In one version, the aberration-correcting dark-field TEM is adapted tooperate in an incoherent illumination mode. In this mode, the coherenceof illumination of the aberration-correcting dark-field TEM is eithersubstantially mitigated or eliminated completely. Incoherence means thatdifferent sets of electrons impinging on the specimen are incoherent inrelation to one another. In one embodiment, the dark-field TEM isimplemented with a substantially incoherent electron source. Forexample, the electron beam source may be adapted to generate an electronbeam having an energy spread of less than about 1 eV. Alternatively orin addition, the dark-field TEM may produce electron beams that areincoherent in relation to one another at different times. For example,the dark-field TEM may differently shift or scan the electron beam overtime. In yet another example, the dark-field TEM spreads the energy ofthe electrons in the beam over different predefined ranges of energiesover time.

By using incoherent illumination, more current can potentially bedirected onto the specimen while simultaneously improving the contrastbetween single heavy atoms or clusters of atoms and a relatively lightatom substrate of the specimen. Incoherent sources can often achievehigher current in exchange for coherence. The contrast improvementarises from the fact that the contrast due to the heavy atom does notdepend on interference of a coherent electron wave whereas the detailsof speckle contrast from the specimen do. Each electron wave thatcontributes to the image will therefore add intensity at the heavy atomposition but average out intensity in the speckle contrast. Thisimprovement may be particularly suited for a system that needs higherdata throughput and less expensive electron sources. In other words, theincoherent illumination mode may enable higher throughput, lessexpensive sources, and better contrast.

The incoherence provides a contrast mechanism that allows directinterpretability of the resulting images. Under incoherent illuminationconditions, phase contrast is reduced whereas amplitude contrast isincreased by the mechanism of superposition: the randomness of imagefeatures in phase contrast signals interferes destructively whereas thescattering from point-like objects sums incoherently. In the aggregate,the scattering information from the point-like objects is retained whilethe phase contrast information from the amorphous substrate isintentionally washed out.

While the incoherent dark-field TEM can be built de novo fromspecifically designed components, there may be practical advantages tomodifying conventional EM systems to provide the advantageouscharacteristics of the inventive systems. For example, such modificationmay allow existing EM facilities to upgrade their current equipment toobtain the advantages of the incoherent dark-field TEM at anadvantageous cost without requiring the construction of an entirely newEM system. The modification may include retrofit of new components andrealignment or repositioning of existing components, such as ofcomponents in the upper column of the EM.

FIG. 9 illustrates an exemplary embodiment of an implementation of anincoherent illumination mode using an incoherent electron source 20A. Afilled cone of many different beams 440 that are incoherent in relationto one another, referred to as incoherently related pencils ofillumination, are emitted by electron source 20A. Each of incoherentlyrelated beams 440 is a constituent that is incoherent in relation to theother constituents. A summation of self-canceling phase contrast noiseand self-reinforcing amplitude signal occurs simultaneously at imageplane 100, so a usable image can be produced at once. Incoherentelectron source 20A may be, for example, an electron source having atungsten or lanthanum hexaboride filament. Incoherent electron source20A may be combined with two or more condenser lenses 450, which can beexcited in different configurations to allow alignment in a coherentillumination mode and then imaging operation in incoherent illuminationmode.

Alternatively or in addition, the incoherent illumination mode may beimplemented in the dark-field TEM without an incoherent electron source.For example, the incoherent illumination mode may be implemented bygenerating electron beams that are incoherent in relation to one anotherat different times. This may be preferable where a conventional EMsystem designed for coherent-mode operation is being modified into oneof the incoherent-mode embodiments described herein.

During exposure of the image, either the angle, the position, or theenergy of the source may be altered. These changes may be made on thetime scale of the exposure. In an illustrative example, if the image isa one-second exposure, any combination of the energy, position, andangle can be oscillated, such as, for example, on the order of about 10times the exposure time. With an incoherent illumination mode, a muchhigher beam current can typically be achieved on the specimen than withcoherent illumination, and therefore imaging may take place much faster.More typically, the exposure time would be, rather than one second, onthe order of milliseconds or microseconds. An advantage of incoherentillumination mode, particularly for the application of DNA sequencing(or any application involving identifying single heavy atoms or clustersof atoms on an otherwise low density background), may be an increase inthe speed of image acquisition that goes with the increased amount ofcurrent that impinges on the specimen.

Either rocking or scanning the electron beam may work as long as therocking or scanning is sufficiently fast. Furthermore, it may suffice toscan or rock the beam within the angular range that is collected by theaperture of the objective lens. This angular range may be considered alimiting factor since, beyond this angular range, the current impingeson the aperture and does not get detected. However, it may be desirableto scan or rock the beam slightly beyond the angular range that iscollected by the aperture of the objective lens, since scattering intothe aperture may nevertheless occur. Nevertheless, rocking or scanningthe beam across an angular range of more than twice the acceptance angleof the objective lens may not be desirable since it would waste current.

A conventional EM system designed to operate in a coherent illuminationmode may be modified into one of the embodiments described herein. ThisEM system may include an optimized system of condenser lenses, such astwo to five condenser lenses. The electron source of the conventional EMsystem may be replaced with an incoherent electron source, such as oneof the incoherent electron sources described herein. In addition,deflectors or other mechanisms may be added for laterally shifting orangularly wobbling the beam, and/or a mechanism may be added to vary theaccelerating voltage applied to the beam. Aberration-correctingillumination systems may be available from, for example, JEOL Ltd. ofTokyo, Japan; FEI Company of Hillsboro, Oreg.; and Carl Zeiss NTS GmbHof Oberkochen, Germany (such as Zeiss's Köhler illumination system). Thetrajectories of the electron rays through one or more of the lenses ofthe illumination system may be altered.

The acceptance angles of the objective lens may be determined based onthe desired resolution of the microscope. For example, if 0.1 nmresolution at 100 kilovolts is desired, one may need approximately 20milliradians acceptance half-angle, and therefore one might preferablynot go beyond 40 milliradians of illumination half-angle. With a greaterangular range, current may undesirably be wasted. In one example,single-atom resolution—namely resolution at least as good as about 0.3nanometers and in some instances at least as good as about 0.15nanometers—may be desirable for a DNA-sequencing application. Once asuitable accelerating voltage is chosen, that resolution requirement maydetermine the acceptance angle of the objective lens.

A conventional EM system whose illumination trajectories have beenmodified to achieve relatively incoherent illumination can be furtherimproved to take advantage of the higher current enabled by incoherentillumination by increasing the speed of the detector or stage. Forexample, a piezoelectric stage may be used. The piezoelectric stage maybe able to move very quickly and settle very quickly and stably so thatshort exposures of the order of milliseconds or microseconds can bepractically achieved. The piezoelectric stage may also be adapted tomove the stage with very high positional precision. Furthermore, thethroughput of data that emerges from the detector, which in this casemay be a high-speed camera, may be quite large, such that electronicscapable of dealing with this data throughput downstream of the cameramay be desirable.

Some of the methods of achieving an incoherent illumination mode includetaking a number of image constituents that are individually coherent andcombining these image constituents incoherently. There are severaldifferent ways of generating and incoherently combining these imageconstituents, as described in more detail below.

FIG. 10 illustrates an exemplary embodiment of a reference version of anADF-TEM that other exemplary embodiments described below will becompared to for the sake of illustration. Illumination 460 is providedparallel to optic axis 130 and onto specimen 40. Beams scattered fromspecimen 40 are collected by the objective lens in objective lens plane470, which focuses the beams onto image plane 100. In back focal plane480 of the objective lens, a diffraction pattern is formed that is theFourier transform of specimen 40, representing angles of scatter fromspecimen 40. Three scattered beams 490 are shown demonstrating that raysscattered to the same angle by different points on specimen 40 convergeto specific points in back focal plane 480 representative of theirscattering angle. In projection, the three points correspond toscattering vectors g, 0 (forward-scattered), and −g. Back focal plane480 contains annular aperture 60, which includes a beam stop 600, acentral disc that leaves an opening 610 with outer diameter D forimplementing the annular-dark-field mode. While the left and rightscattered beams pass through annular aperture 60, the forward-scatteredbeam (0) is blocked by beam stop 500 in back focal plane 480. Only therays passing through annular aperture 60 propagate to image plane 100,forming a dark-field image.

There is a relationship between the aperture diameter D and resolution.Aperture 60 selects the range of angles that are used to form the imageor probe in TEM or STEM, respectively. In the case of TEM, specimen 40is illuminated and electrons are scattered from different points onspecimen 40. The electrons scattered to a particular angle fromdifferent parts of specimen 40 are brought to a common point in backfocal plane 480, and then propagated further until they form an image.Aperture 60 thus selects the angles which form the image by limiting therays passing through back focal plane 480. In the case of STEM, where asource produces a plane wave that enters the objective lens, theobjective aperture placed in back focal plane 480 limits the size ofillumination entering the objective lens. The rays that the objectivelens focuses to a point on the specimen are thus limited in angle by theaperture.

FIG. 11 illustrates a schematic diagram of an exemplary embodiment of animplementation in which an electron beam 620 is tilted at an angle inrelation to optic axis 130 of the EM column. Electron beam 620 may bescanned radially such that electron beam 620 keeps substantially thesame angle in relation to optic axis 130, forming a substantiallycylindrically symmetric cone of electron illumination about optic axis130. Alternatively, the angle of tilted electron beam 620 may be flippedbetween two angles that are symmetric (or “mirror” angles) with respectto optic axis 130, the figure showing an example of one of the twomirror angles. For example, electron beam 620 may be passed through atilting prism that is used to alternately flip electron beam 620 betweenthese two mirror angles. Specimen 40 scatters the incident electrons,resulting in scattered beams 630, shown in the figure as two beams onthe sides and the one beam in the middle. Scattered beams 630 arefocused by the objective lens to the image plane. Scattered beams 630create a diffraction pattern in back focal plane 480 and are filtered byaperture 633 with a circular disc opening 637. Image constituents fromdifferent scan positions of electron beam 620 are summed. These imageconstituents are incoherent in relation to one another. By illuminatingspecimen 40 through a cone of illumination, or alternately illuminatingspecimen 40 at mirror angles, and collecting these image contributionson image plane 100 over time, an incoherently-summed image can beproduced. The tilt angle may be less than about 100 milliradians inrelation to optic axis 130 and may exceed the aperture radius D/2.

As shown in FIG. 11, these embodiments may be implemented in dark-fieldmode without annular aperture 60 having central beam stop 600. As shownin the figure, aperture 633 may be adapted, and the tilt angle may beselected, to be sufficiently high, to cause the zero beam to impinge onaperture 633 and not pass through opening 637 in back focal plane 480.While the right scattered beam pass through aperture 633, theforward-scattered beam (0) and left scattered beam are blocked byaperture 633 in back focal plane 480. Only the rays passing throughopening 637 propagate to image plane 100, forming a dark-field image.Alternatively, an annular aperture, such as shown in FIG. 10, may beused in place of aperture 633 with circular disc opening 637.

FIG. 12 illustrates a generalized version of the manner in whichamplitude contrast is enhanced in incoherent annular-dark-field imagingwhile phase contrast is decreased to improve contrast of point-likeobjects in the specimen. This figure shows an ideal amplitude signal 640from a specimen containing a point-like object on an amorphousbackground. For the sake of illustration, different incoherently relatedimage constituents, labeled as 650, 660, 670, 680, etc., that arecreated from the object are shown as vertically arranged. For each ofthese image constituents, the horizontal axis represents position andthe vertical axis represents signal amplitude. Each of the imageconstituents contains an amplitude contrast component and also a phasecontrast component. The latter component dominates in TEM images, as canbe seen in image constituents 650, 660, 670, 680, etc. The methodsdescribed herein extract amplitude signal 640 from the phase contrastnoise.

For example, a number of image constituents 650, 660, 670, 680, etc.,are taken either in sequence or simultaneously in a particular imagingmode. In each of image constituents 650, 660, 670, 680, etc., theamplitude signal is small in comparison to the speckle noise from thephase contrast. However, the speckle noise varies substantially betweendifferent image constituents 650, 660, 670, 680, etc. Meanwhile, hiddenwithin this noise is a weak yet consistent amplitude signal 640 betweendifferent image constituents 650, 660, 670, 680, etc. Thus, when imageconstituents 650, 660, 670, 680, etc., are superimposed, the phasecontrast noise tends to cancel while amplitude signal 640 reinforces,forming an increasingly discernible signal 690 as moreincoherently-related constituent images are included.

FIG. 13 illustrates an example of yet another embodiment of theimplementation of incoherent superposition. In this figure, differentenergies are used such that the scattering angles change slightly. Sincethe objective lens focuses higher energy electrons less strongly thanlower energy electrons, the scattering angles from the specimen varyaccordingly, as indicated in the figure by scattered beams 490 and 700,representing, respectively, lower and higher energy beams. In thisfigure, in contrast to FIG. 10, part of the beam that would have beenimaged as a point in image plane 100 is now imaged onto a diffuse region710 and another part of beam 460 that would have been imaged onto apoint is now imaged onto a narrower diffuse region 720. While regions710 and 720 represent diffuse images of the same point of specimen 40,their centers are still approximately in the same location whenprojected onto image plane 100. A point-like amplitude object, whenimaged in this way, will consistently be imaged to the same point.Simultaneously, speckle contrast from the background will be averagedout.

Adjustment of electron energy can be achieved by various alternativemethods including, for example, choosing a source with a large energyspread in the electron source, increasing the chromatic aberration inthe illumination system, and modulating the voltage applied to theelectron source with time at a frequency greater than the exposurefrequency.

FIG. 14 illustrates an example of yet another embodiment of theimplementation of incoherent superposition. In this embodiment, imagesexposed at different times are summed. Again, through the summation,more amplitude contrast is generated while the phase contrast isdecreased.

FIG. 15 illustrates still another exemplary embodiment of theimplementation of incoherent superposition. In FIG. 15, the electronbeam is shifted laterally to different lateral positions, such asposition 725 and position 730, at different times to obtain differentsets of scattered beams 490 and 495 and the resulting differentrelatively incoherent constituents. This shift can be achieved in oneexample by using dipole deflectors to shift the beam before it reachesthe sample.

FIG. 16 illustrates yet another embodiment in which a prism 740 is usedin the beam path to shift all or a portion of the beam, such as fromposition 725 to position 730.

The examples shown in FIGS. 11, 13, 14, 15, and 16 illustrate variousembodiments in which constituent images are exposed serially or inparallel to improve amplitude contrast relative to phase contrast,thereby improving interpretability of the image.

The electron energy used in the dark-field TEM may be determined atleast in part based on the transmission properties of the specimen. Thespecimen may have a thickness on the order of 2 nanometers, such as forexample a thickness of about 1 nanometer. In one example, the specimenis made of carbon, although single-atom-thick graphene may also be used.As a result, 1 keV electrons are likely to be the lowest energyappropriate when considering voltage alone.

Unfortunately, since electron wavelength varies inversely with energy,the diffraction limit may require the angles to be corrected to belarge. Such an aberration-correcting dark-field TEM may becomechallenging to manufacture. It may be desirable for the dark-field TEMto be operated at a much higher voltage, such as from about 1 kV toabout 300 kV. For example, the dark-field TEM may be operated at about30 kV. This voltage range is in the realm of conventional microscopy,and the implementation of electrostatic correction elements may beunfeasible in this range due to the risk of damage from high localfields, high voltage discharge, and transmission of high-energyelectrons.

For miniaturized embodiments of the aberration-correcting dark-field TEMcolumn, the voltage may be based in part on the dimensions of theminiaturized embodiments. The miniaturization of the column could goeven further than described above. Such a miniaturizedaberration-correcting dark-field TEM column remains a column withaberration correction by charge-on-axis elements with substantially thesame features described herein. A specialized detector may also beuseful so that operation of the instrument is in STEM mode or SEM mode,rather than in TEM mode. In that case, a fabricated solid-statebackscatter detector may be provided.

The dark-field TEM may preferably use a beam current of from about 10picoamps to about 1 milliamp. At beam currents above around 100microamps, coherence decreases. When an incoherent illumination mode isintended, the high spatial charge density may desirably increase theincoherence. Thus, for an incoherent illumination mode, a beam currentabove 100 microamps may be used advantageously.

Furthermore, it may be desirable for the beam to be sufficientlymonochromatic, in other words to have a sufficiently narrow range ofenergies, to avoid focus problems. A spread in energies of the electronsin the beam typically causes a corresponding change of focus of theimage. Thus, the image may be thought of as a sum of many images thathave changing foci. If that range is too large, then the intensity of asingle atom in the image may get blurred out over a large region andthereby become indistinguishable from the background. Thus, it may bepreferable to have an energy spread of less than about 10 eV to avoidsuch blurring. Where a tighter focus is desired, however, it may bepreferable to have an energy spread of less than about 1 eV. Forexample, the electron beam may even have an energy spread of less thanabout 200 meV. This may be desirable where there is no chromaticaberration correction in the optical system. In other circumstances,however, such as if chromatic-aberration correction is implemented inthe optical system, much larger energy spreads may be used. For example,the chromatic-aberration correctors described herein may be able tohandle hundreds of electron volts of energy spread.

Although the foregoing embodiments have been described in detail by wayof illustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art in lightof the description herein that certain changes and modifications may bemade thereto without departing from the spirit or scope of the appendedclaims. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only,” and the like in connection with therecitation of claim elements, or use of a “negative” limitation. As willbe apparent to those of ordinary skill in the art upon reading thisdisclosure, each of the individual aspects described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalaspects without departing from the scope or spirit of the disclosure.Any recited method can be carried out in the order of events recited orin any other order which is logically possible. Accordingly, thepreceding merely provides illustrative examples. It will be appreciatedthat those of ordinary skill in the art will be able to devise variousarrangements which, although not explicitly described or shown herein,embody the principles of the disclosure and are included within itsspirit and scope.

Furthermore, all examples and conditional language recited herein areprincipally intended to aid the reader in understanding the principlesof the invention and the concepts contributed by the inventors tofurthering the art, and are to be construed without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles and aspects of the invention, as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryconfigurations shown and described herein. Rather, the scope and spiritof present invention is embodied by the claims.

In this specification, various preferred embodiments have been describedwith reference to the accompanying drawings. It will be evident,however, that various other modifications and changes may be madethereto and additional embodiments may be implemented, without departingfrom the broader scope of the claims that follow. The specification anddrawings are accordingly to be regarded in an illustrative rather thanrestrictive sense.

We claim:
 1. A transmission electron microscope comprising: an electronbeam source to generate an electron beam; beam optics to converge theelectron beam, the beam optics defining an optic axis of the microscopealong which there is substantial cylindrical symmetry of the beamoptics; an aberration corrector comprising a foil located approximatelyat the optic axis, the aberration corrector being adapted to correct theelectron beam for at least a spherical aberration; a specimen holder tohold a specimen in the path of the electron beam; and a detector todetect the electron beam transmitted through the specimen.
 2. Thetransmission electron microscope of claim 1, wherein the foil comprisesa conductive grid, the conductive grid comprising a metal.
 3. Thetransmission electron microscope of claim 2, wherein the conductive gridhas a thickness of from about 0.1 nm to about 10 nm.
 4. The transmissionelectron microscope of claim 1, wherein the foil comprises a materialthat changes at least one dimension in relation to temperature, andfurther comprising a temperature applicator to change the temperature ofthe foil to induce a change in an electric field created by the foil. 5.The transmission electron microscope of claim 1, wherein the foilcomprises (i) two metal layers and (ii) an insulating layer between thetwo metal layers, and wherein the foil has an aperture passing throughthe metal layers and the insulating layer.
 6. The transmission electronmicroscope of claim 5, wherein the foil further comprises a filmcomprising carbon on one of the metal layers, on the side of the metallayer that is opposite the insulating layer.
 7. The transmissionelectron microscope of claim 1, wherein the aberration corrector isadapted to correct for a parasitic aberration.
 8. The transmissionelectron microscope of claim 7, wherein the aberration corrector furthercomprises a multipole element to (i) predistort the electron beam beforethe electron beam enters the foil and (ii) undistort the electron beamafter the electron beam has left the foil.
 9. The electron beam assemblyof claim 1, comprising an annular aperture to produce a dark-field modein which a zero beam of the electron beam is not detected, thedark-field stop comprising (i) a circular-disc stop with a substantiallycircular cross-section approximately at the radial center of theelectron beam to stop a central portion of the electron beam; and (ii)an outer stop concentric with, and spaced from, the circular-disc stop,such that there is an annular gap between the circular-disc stop and theouter stop.
 10. The electron beam assembly of claim 9, wherein theannular aperture is adapted to limit transmission of electrons scatteredfrom the specimen to an angular range between an inner angle and anouter angle, the inner angle being from about 0.1 mrad to about 20 mrad,and the outer angle being at least about 1 mrad.
 11. The transmissionelectron microscope of claim 1, wherein the beam optics comprise anobjective lens, and wherein the foil is positioned outside the objectivelens.
 12. A transmission electron microscope comprising: an electronbeam source to generate an electron beam; beam optics to converge theelectron beam, the beam optics defining an optic axis of the microscopealong which there is substantial cylindrical symmetry of the beamoptics; an aberration corrector comprising a set of elementsapproximately concentric about the optic axis, the concentric elementsbeing adapted to have different respective electric potentials appliedto them, the aberration corrector being adapted to correct the electronbeam for at least a spherical aberration; a specimen holder to hold aspecimen in the path of the electron beam; and a detector to detect theelectron beam transmitted through the specimen.
 13. The transmissionelectron microscope of claim 12, wherein at least one of the concentricelements is approximately cylindrically symmetric.
 14. The transmissionelectron microscope of claim 13, wherein the cylindrically symmetricconcentric element has the shape of a conical section.
 15. Thetransmission electron microscope of claim 13, wherein the cylindricallysymmetric concentric element has a curved surface.
 16. The transmissionelectron microscope of claim 13, wherein the set of elements of theaberration corrector comprises at least three elements, the threeelements being adapted to have different respective electric potentialsapplied to them.
 17. The transmission electron microscope of claim 13,wherein the cylindrically symmetric concentric element is a ring, andwherein the aberration corrector further comprises two or more spokes tosupport the ring.
 18. The transmission electron microscope of claim 12,wherein the aberration corrector is adapted to correct for a parasiticaberration.
 19. The transmission electron microscope of claim 12,wherein the beam optics comprise an objective lens, and wherein the setof elements of the aberration corrector is positioned outside theobjective lens.
 20. A method for diagnosing aberrations in atransmission electron microscope that is adapted to operate in adark-field mode in which a zero beam of the electron beam is notdetected, the method comprising: acquiring one or more images, as afunction of either illumination tilt or defocus, from the transmissionelectron microscope; extracting a value for the blurring effect of thetilt or defocus from the images; and calculating a value for defocus orastigmatism based on the value for the blurring effect and the value foreither the illumination tilt or the defocus.